The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art in the present invention.
Nucleic acid ligases belong to a class of enzymes that catalyze phosphodiester bond formation between adjacent 3′-hydroxyl and 5′-phosphoryl termini in nucleic acid (e.g., RNA or DNA) in the presence of a cofactor, such as ATP or NAD+. Ligases are employed in a number of molecular biology applications including nucleic acid sequence detection, single nucleotide polymorphism (SNP) detection, protein detection, sequencing by ligation, and ligase chain reaction (LCR).
In biochemical fidelity experiments, DNA ligases have been found to tolerate a variety of nucleic acid substrate mismatches. For example, T4 DNA ligase has a tolerance for mismatches that results in a propensity to seal one of every 103 mismatched duplexes. Showalter, A. K., et al., 106 Chem. Rev, 340-360 (2006). In comparison, the error rate of a conventional DNA polymerase is approximately one error for every 105-106 dNTP insertions, several orders of magnitude higher in fidelity than ligase. Other atypical joining reactions of DNA ligase include intramolecular loop formation (Western, L., et al., 19 Nucleic Acids Res, 809-813 (1991)), joining of non-overlapping, blunt-ended duplexes (Barringer, K., et al., 89 Gene, 117-122 (1990), Cao, W., 22 Trends Biotechnol., 38-44 (2004)) and template-independent reactions (Barringer, K., et al., Kuhn, H., et al., 272 FEBS J, 5991-6000 (2005)).
Various approaches have been described for improving DNA ligation fidelity. For example, Luo, J., et al., 24 Nucleic Acids Res, 3079-3085 (1996) disclose modifying the third nucleotide upstream from the 3′-OH, acceptor with universal base 3-nitropyrrole and site directed mutagenesis of the ligase protein. Tong, J., et al., 27 Nucleic Acids Res, 788-794 (1999); Feng, H., et al., 43 Biochemistry, 12648-12659 (2004); Jeon, H., et al., 237 FEMS Microbiol Lett., 111-118 (2004); Lim, J., et al., 388 Arch Biochem Biophys., 253-260 (2001); and Luo, J., et al., 24 Nucleic Acids Res, 3071-3078 (1996) disclose mutating amino acid residues in the DNA ligase. Cao, W., 22 Trends Biotechnol., 38-44 (2004) disclose using an endonuclease in the ligation reaction. Egholm, M., et al., U.S. Pat. No. 6,297,016 disclose acceptor modifications. Fung, S., et al., U.S. Pat. No. 5,593,826 discloses 3′-NH2 substituted acceptor probes. Bandaru, R., et al., U.S. Pat. Nos. 6,811,986 and 6,635,425 discloses use of 5′-thiophosphates in the donor (5′-phosphate) strand.
Modified ligase cofactors have been used determine ligase cofactor dependence and as ligation inhibitors. See e.g., Montecucco, A., et al., 271 Biochem J., 265-268 (1990); Shuman, S., 34 Biochemistry, 16138-16147 (1995); Raae, A., et al., 81 Biochem. Biophys. Res. Commun., 24-27 (1978); Cherepanov, A. V., et al., 269 Eur. J. Biochem., 5993-5999 (2002); Belford, H. G., et al., 268 J Biol Chem, 2444-2450 (1993); Doherty, A. J., et al., 271 J Biol Chem, 11083-11089 (1996); Ho, C. K., et al., 71 J Virol, 1931-1937 (1997); Lai, X., et al., 6 Extremophiles, 469-477 (2002); and Sriskanda, V., et al., 28 Nucleic Acids Res, 2221-2228 (2000).
Nucleic acid library preparation schemes involve the addition of adapter sequences onto the 5′- and 3′-termini of target nucleic acids. For RNA libraries, adapters are typically added in two sequential ligation steps to minimize adapter dimer formation, as described in Tian, G., et. al., 10 BMC Biotechnol, 64 (2010). First, an adenylated version of the 3′-adapter probe is ligated onto the 3′-terminus of the RNA library, in the absence of ATP, using a truncated version of T4 RNA ligase 2 that utilizes 5′-adenylated, rather than 5′-phosphorylated probes. Next, the 5′-adapter probe sequence is added onto the 5′-end of the RNA library using T4 RNA ligase 1. One approach to suppress adapter dimer formation is by hybridization of the cDNA synthesis primer after ligation of the 3′-adapter and before the 5′-adapter ligation step, as described in Nakashe, P., et al. 1 Journal OMICS Research, 6-11 (2011). Another approach, described by Kawano, M., et al. 49 Biotechniques, 751-755 (2010) employs a hybridization step between adapter dimers and an LNA sequence to block downstream replication.
Several approaches to tag double-stranded DNA (dsDNA) libraries in preparation for next generation sequencing (NGS) have been described, as reviewed by Linnarsson, S., 316, Exp Cell Res 1339-1343 (2010), and most involve a ligation step. In one approach, probe sequences are added onto dsDNA libraries by ligation of a pair of 5′-phosphorylated blunt-ended dsDNA adapter probes (P1 and P2) onto 5′-phosphorylated blunt-ended dsDNA libraries. The P1 and P2 adapter sequences are added onto both ends of the dsDNA library using T4 DNA ligase in a single step. Blunt ended adapter ligation strategies are typical for SOLiD (Life Technologies, Carlsbad, Calif.) and 454 GS FLX (Roche, Branford, Conn.) workflows. In another approach, the dsDNA library is subjected to a different polishing step, which produces dsDNA libraries that contain a single A tail on the 3′-end. The A-tailed dsDNA library is ligated to a dsDNA adapter, which contains a single T tail on the 3′-terminus and a 5′-phosphate in a single step using T4 DNA ligase. A-tailed ligation strategies are typical for the Genome Analyzer platform (Illumina, San Diego, Calif.). All three approaches are prone to adapter dimer formation, as described by Linnarsson, S., 316, Exp Cell Res 1339-1343 (2010); Quail, M. A., et al. 5, Nat Methods 1005-1010 (2008), and Huang, J., et al., 6, PLoS One, e19723 (2011).